Protein tyrosine phosphatase N2 regulates TNF alpha- induced signalling and cytokine secretion in human intestinal epithelial cells

ArticleinGut 60(2):189-97 · February 2011with18 Reads
DOI: 10.1136/gut.2010.216606 · Source: PubMed
The Crohn's disease (CD) susceptibility gene, protein tyrosine phosphatase N2 (PTPN2), regulates interferon γ (IFNγ)-induced signalling and epithelial barrier function in T₈₄ intestinal epithelial cells (IECs). The aim of this study was to investigate whether PTPN2 is also regulated by tumour necrosis factor α (TNFα) and if PTPN2 controls TNFα-induced signalling and effects in IECs. T₈₄ IECs were used for all cell studies. Protein levels were assessed by western blotting, mRNA levels by reverse transcription-PCR (RT-PCR) and cytokine levels by ELISA. PTPN2 knock-down was induced by small interfering RNA (siRNA). Imaging was performed by immunohistochemistry or immunofluorescence. TNFα treatment elevated PTPN2 mRNA as well as nuclear and cytoplasmic protein levels and caused cytoplasmic accumulation of PTPN2. Biopsy specimens from patients with active CD showed strong immunohistochemical PTPN2 staining in the epithelium, whereas samples from patients with CD in remission featured PTPN2 levels similar to controls without inflammatory bowel disease (IBD). Though samples from patients with active ulcerative colitis (UC) revealed more PTPN2 protein than non-IBD patients and patients with UC in remission, their PTPN2 expression was lower than in active CD. Samples from patients with CD in remission and responding to anti-TNF treatment also showed PTPN2 levels that were similar to those in control patients. Pharmacological inhibition of nuclear factor-κB (NF-κB) by BMS-345541 prevented the TNFα-induced rise in PTPN2 protein, independent of apoptotic events. PTPN2 knock-down revealed that the phosphatase regulates TNFα-induced extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 phosphorylation, without affecting c-Jun N-terminal kinase (JNK), inhibitor of κB (IκB) or NF-κB phosphorylation. Loss of PTPN2 potentiated TNFα-induced secretion of interleukin 6 (IL-6) and IL-8. In TNFα- and IFNγ-co-treated cells, loss of PTPN2 enhanced protein expression of inducible nitric oxide synthase (iNOS). TNFα induces PTPN2 expression in IECs. Loss of PTPN2 promotes TNFα-induced mitogen-activated protein kinase signalling and the induction of inflammatory mediators. These data indicate that PTPN2 activity could play a crucial role in the establishment of chronic inflammatory conditions in the intestine, such as CD.
    • "It is interesting to note that Ptpn2+ mice display greater susceptibility to DSS than wild-type Ptpn2 mice, suggesting that Ptpn2 negatively regulates inflammation (103). Expression of PTPN2 in human T84 colonocytes is enhanced by IFN-γ and TNF-α and accordingly PTPN2 mRNA levels are increased in the inflamed mucosa of patients with CD (104, 105). Since the link between defective autophagy and CD was identified, a potential role for PTPN2 in the regulation of autophagy has also been investigated. "
    [Show abstract] [Hide abstract] ABSTRACT: One of the most significant challenges of cell biology is to understand how each type of cell copes with its specific workload without suffering damage. Among the most intriguing questions concerns intestinal epithelial cells in mammals; these cells act as a barrier between the internally protected region and the external environment that is exposed constantly to food and microbes. A major process involved in the processing of microbes is autophagy. In the intestine, through multiple, complex signaling pathways, autophagy including macroautophagy and xenophagy is pivotal in mounting appropriate intestinal immune responses and anti-microbial protection. Dysfunctional autophagy mechanism leads to chronic intestinal inflammation, such as inflammatory bowel disease (IBD). Studies involving a number of in vitro and in vivo mouse models in addition to human clinical studies have revealed a detailed role for autophagy in the generation of chronic intestinal inflammation. A number of genome-wide association studies identified roles for numerous autophagy genes in IBD, especially in Crohn's disease. In this review, we will explore in detail the latest research linking autophagy to intestinal homeostasis and how alterations in autophagy pathways lead to intestinal inflammation.
    Full-text · Article · Sep 2013
    • "Several phosphorylated proteins are well known targets of dephosphorylation by PTPN2, including the epidermal growth factor receptor [11], vascular endothelial growth factor receptor-2 [12], the insulin receptor [13], signal transducers and activators of transcription 1 and 3 (STAT1 and STAT3) [14], [15], and mitogen-activated protein kinase (MAPK)-isoforms, such as p38 [16]. Inactivation of those substrates by dephosphorylation results in the negative regulation of signaling pathways involved in inflammatory responses induced by the pro-inflammatory cytokines IFN-γ [17], [18], TNF [19] and interleukin (IL)-6 [14]. "
    [Show abstract] [Hide abstract] ABSTRACT: Spermidine is a dietary polyamine that is able to activate protein tyrosine phosphatase non-receptor type 2 (PTPN2). As PTPN2 is known to be a negative regulator of interferon-gamma (IFN-γ)-induced responses, and IFN-γ stimulation of immune cells is a critical process in the immunopathology of inflammatory bowel disease (IBD), we wished to explore the potential of spermidine for reducing pro-inflammatory effects in vitro and in vivo. Human THP-1 monocytes were treated with IFN-γ and/or spermidine. Protein expression and phosphorylation were analyzed by Western blot, cytokine expression by quantitative-PCR, and cytokine secretion by ELISA. Colitis was induced in mice by dextran sodium sulfate (DSS) administration. Disease severity was assessed by recording body weight, colonoscopy and histology. Spermidine increased expression and activity of PTPN2 in THP-1 monocytes and reduced IFN-γ-induced phosphorylation of signal transducer and activator of transcription (STAT) 1 and 3, as well as p38 mitogen-activated protein kinase (MAPK) in a PTPN2 dependent manner. Subsequently, IFN-γ-induced expression/secretion of intracellular cell adhesion molecule (ICAM)-1 mRNA, monocyte chemoattractant protein (MCP)-1, and interleukin (IL)-6 was reduced in spermidine-treated cells. The latter effects were absent in PTPN2-knockdown cells. In mice with DSS-induced colitis, spermidine treatment resulted in ameliorated weight loss and decreased mucosal damage indicating reduced disease severity. Activation of PTPN2 by spermidine ameliorates IFN-γ-induced inflammatory responses in THP-1 cells. Furthermore, spermidine treatment significantly reduces disease severity in mice with DSS-induced colitis; hence, spermidine supplementation and subsequent PTPN2 activation may be helpful in the treatment of chronic intestinal inflammation such as IBD.
    Full-text · Article · Sep 2013
    • "The formation and maintenance of an intact intestinal barrier is critical to maintain normal physiological function and prevent diseases, such as ulcerative colitis, Crohn disease, chronic diarrhoeal disease and other infectious and inflammatory intestinal diseases (Meddings et al. 1994). Significant data suggest that TNF-α is involved in the dysregulation of intestinal EBF (Lorenzo-Z ´ uñiga et al. 2006; Kolodziej et al. 2011; Marchiando et al. 2011; Noth et al. 2011; Scharl et al. 2011; Ye et al. 2011) and is a critical pathogenic component of many related diseases. Tumour necrosis factor-α and other pro-inflammatory factors, such as interferon-γ, are major factors which contribute to TPN-associated loss of EBF. "
    [Show abstract] [Hide abstract] ABSTRACT: OBJECTIVE:TNF-α has been reported to play a central role in intestinal barrier dysfunction in many diseases; however, the precise role of the TNF-a receptors (TNFRs) using in vivo models has not been well-defined. Our previous data showed that enteral nutrient deprivation or total parenteral nutrition (TPN) led to a loss of intestinal epithelial barrier function (EBF), with an associated up-regulation of TNF-α and TNFR1. In this study, we hypothesized that TNF-α played an important role in TPN-associated EBF dysfunction. Using a mouse TPN model we explored the relative roles that TNFR1 versus TNFR2 had in mediating this barrier loss.DESIGN:C57/BL6 mice underwent intravenous cannulation and were given enteral nutrition or TPN for 7 days. TNF-α receptor knockout (KO) mice, including TNFR1-KO, R2-KO, or R1R2-double KO (DKO), were used. Outcomes included small intestine transepithelial resistance (TER) and tracer permeability, junctional protein ZO-1, occludin, claudins and E-cadherin expression. To further address the dependence of TNF-α on EBF dysfunction, exogenous TNF-α and pharmacological blockade of TNF-α (Etanercept) were also performed.RESULTS:TPN led to a loss of EBF, and this was almost completely prevented in TNFR1R2DKO mice, and partially prevented in TNFR1KO mice, but not in TNFR2KO mice. TPN associated down-regulation of junctional protein expression and junctional assembly was almost completed prevented in the TNFR1R2DKO group. Blockade of TNF-α also prevented EBF and junctional protein losses in TPN mice. TPN administration up-regulated the downstream NF-кB and MLCK signaling, and these changes were almost completely prevented in TNFR1R2DKO mice, as well as with TNF-α blockade, but not in TNFR1KO or TNFR2KO TPN groups.CONCLUSIONS:TNF-α is a critical factor for TPN-associated epithelial barrier dysfunction; and both TNFR1 and TNFR2 receptors are involved in EBF loss. NF-кB and MLCK signaling appear to be important downstream mediators involved in this TNF-α signaling process.
    Article · Jun 2013
Show more